mRNA control permeates biology. Many transcripts contain cis-acting regulatory features in their 3' untranslated regions (3'UTRs) 1. These elements recruit regulatory factors capable of modulating transport, translation, and stability 2,3. Multiple RNA-binding proteins congregate onto a given 3'UTR; often interacting directly with one another 5. Such complexes are a dominant theme in mRNA control particularly during early development 6,7. Our ultimate aim is to characterize the molecular and structural mechanisms promoting assembly of regulatory proteins onto the 3'UTR. The core theme of this proposal is to elucidate key aspects of these complexes by focusing on a pair of interacting proteins implicated in diverse biological processes spanning early development, learning, and memory 8-13. We have chosen to study the interaction between a CPEB (Cytoplasmic Polyadenylation Element Binding) and PUF (Pumilio and FBF) protein. Members of these two families collaborate to regulate mRNA expression via binding to the 3' UTR 14. The hypothesis underlying much of this work is that the interaction between PUF and CPEB proteins is mediated by a discrete molecular interface. My preliminary data support this idea: a 40 amino acid peptide in CPB-1 and a short loop in FBF-2 are required for their interaction. In the first aim, we analyze the molecular basis of the interaction in depth, isolating mutations that disrupt or enhance binding using assays developed in the Wickens lab 35. In the second aim, we test the hypothesis that the binding affinity of FBF-2 for mRNA is enhanced by CPB-1. To do so, we use a high-throughput sequencing strategy analyzing FBF-2 sequence specificity with and without CPB-1. In the third aim, we test the hypothesis that the CPB-1/FBF interaction enhances transational repression, and is required for spermatogenesis in vivo. We build on preliminary data that suggest CPB-1 enhances repression by FBF-2 in vitro. We also describe a new assay in which we disrupt the complex by injection of short peptides into living animals. My specific aims are as follows. Aim 1 - To identify residues in FBF-2 and CPB-1 required for their interaction. Aim 2 - To determine the effects of CPB-1 binding on the affinity of FBF-2 for RNA. Aim 3 - To elucidate the functional effects of CPB-1 binding to FBF-2. The depth in which we will study a 3'UTR complex is innovative as are the techniques we use; including deep sequencing to assess RNA binding specificity, and a peptide injection strategy to assess function in vivo. Our research, while focused tightly on the PUF-CPEB interaction, will bear broadly on mechanisms of 3'UTR control.